Can Tho Journal of Medicine and Pharmacy 9(6) (2023)
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PHYTOCONSTITUENTS AND IN VITRO ANTI-UROLITHIATIC
ACTIVITY OF VARIOUS HYDROPHILIC EXTRACTS OF
TERMINALIA CATAPPA LEAVES ON CALCIUM OXALATE CRYSTAL
Vo Dang Thuan, Huynh Anh Duy*
College of Natural Sciences, Can Tho University
*Corresponding author: haduy@ctu.edu.vn
Received: 13/5/2023
Reviewed: 25/5/2023
Accepted: 19/09/2023
ABSTRACT
Background: Medicinal plants play an important role in the alternative or complement
therapy to manage of urinary stones at this time. Terminalia genus was proved anti-urolithiatic activity
via in vitro inhibition of calcium oxalate formation. Among the samples, Terminalia catappa showed
as a potential plants for this activity in India. Moreover, they were a common plant species in Vietnam
and there was no research on this topic in our country. Objectives: To evaluate in vitro anti-
urolithiatic activity of Terminalia catappa leaves in Vietnam, through inhibition of calcium oxalate
formation. In addition, moisture value, preliminary screening of the chemical composition and
determination of tanninoid content of aqueous extract also were conducted. Materials and methods:
Moisture content of herbs was conducted according to guidelines of 5th Vietnam pharmacopoeia,
appendix 9.6. Qualitation of the phytochemical constituents of aqueous extract with appropriate
reagents to confirm the presence of natural compounds via chemical reactions. Determination of
tanninoid content of aqueous extract was evaluated by using oxidation-reduction titration method
(Lowenthal assay) and skin powder method in instructing of appendix 12.6 from 5th Vietnam
pharmacopoeia. Inhibitory effect of calcium oxalate formation of three hydrophilic extracts (45%
ethanol, 96% ethanol and aqueous extracts) was confirmed by nucleation assay with cystone as a
positive control. Results: Moisture content of medicinal plant was 11.625%. Terminalia catappa
leaves contain the major phytochemical constituents such as flavonoids, tanninoids and saponins. The
tanninoid content according to Lowenthal method and skin powder method were 10.88% and 10.70%,
respectively. Therefore, the average tannin content was confirmed to be approximately 10.79%.
Among the investigated samples, aqueous extract showed the best inhibitory activity of calcium oxalate
crystal formation with an IC50 of 602.67 μg/mL when compared to cystone with IC50 423.05 μg/mL.
Conclusions: The aqueous extract from Terminalia catappa leaves has been shown to be a promising
source for anti-calcium oxalate crystals formation activity on experimental model. The anti-urolithic
potential of Terminalia catappa leaves may be related to its major phytoconstituents.
Keywords: Terminalia catappa, tanninoid, nucleation assay, calcium oxalate.
I. INTRODUCTION
Kidney stones (nephrolithiasis) were formed by the deposition of crystals in the
kidney. Urinary stone (urolithiasis) was a condition that occurs when these stones escaped
the kidney and moved into other part of the urinary system, including the ureters, bladder,
and urethra. These stones have been associated with urinary tract obstruction, kidney failure,
and urinary infections [1]. According to an epidemiological survey of urolithiasis in Asia,
the prevalence rate was 5-19.1% in West Asia, Southeast Asia, South Asia and some
developed countries (Korea and Japan). Meanwhile, this percentage was only 1-8% in most
of East Asia and North Asia. In addition, calcium oxalate was the most common kidney
Can Tho Journal of Medicine and Pharmacy 9(6) (2023)
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stone composition, accounting for 75-90%, followed by uric acid (5-20%), calcium
phosphate (6-13%), struvite (2-15%), apatite (1%) and cystine (0.5-1%) [2]. In Vietnam,
the estimated prevalence rate of urolithiasis ranged from 2-12%. According to a recent
report, this percentage shows an upward trend according to the data from 1990 to 2019 [3].
Data from in vitro, in vivo and clinical trials suggested that herbs may be considered
as an alternative or adjunct therapy in urolithiasis treatment [4]. In India, species of the
genus Terminalia have been demonstrated to be a promising medicinal product for
urolithiasis treatment through in vitro screening data [5]. Based on the above information,
this study aims to evaluate the in vitro inhibitory effect against urinary stones on an anti-
calcium oxalate crystal formation model and phytochemical data of Terminalia catappa
leaves, a species of the genus Terminalia in Vietnam.
II. MATERIALS AND METHODS
2.1. Materials
Terminalia catappa leaves, collected in August 2021 in Ninh Kieu district, Can Tho
city, Vietnam. Fresh leaves (3 kg) were thoroughly washed, damaged parts were removed.
After that, the samples were left to dry, then later dehydrated at 60 °C until dry. The samples
were then ground to a fine powder and used for further assay.
2.2. Methods
2.2.1. Preliminary phytochemical evaluation of Terminalia catappa leaves
Determination of the moisture
According to the instructions in Vietnamese Pharmacopoeia V, Appendix 9.6. [6]
Preliminary identification of phytochemical components
Qualitative analysis to identify alkaloids, flavonoids, anthocyanins,
proanthocyanidins, tanninoids, saponins according to literature [7].
Tanninoid quantification
Oxidation-reduction titration (Lowenthal assay)
The experiment was conducted according to Wangiyana et al (2019) [8]. Take 5 mL
of the sample solution into a conical flask, add 150 mL of distilled water, 5 mL of indigo
carmine solution, and then quantify with KMnO4, drip and shake well until a yellow color
appears, then stop. Simultaneously, quantify a blank sample with 5 mL of indigo carmine
solution dissolved in 150 mL of water. 1 mL of KMnO4 equivalent to 4,157 mg of tannins.
The tanninoid content was calculated using the formula (2.1):
X: tannin content, calculated as dry herbs (%); a: mean volume of KMnO4 in sample
titration (mL); b: mean volume of KMnO4 in blank titration (mL); V1: volume of sample
solution to be quantified (10 mL); V2: volume of the flask (250 mL); G: Weight of the herbs,
minus moisture (g)
Skin powder method
The experiment was conducted according to Vietnam Pharmacopoeia V, Appendix
12.6 [6]. Three samples were weighed and recorded mass as T1, T2 and To (gram),
respectively. The percentage (%) of tanninoids was calculated using the formula (2.2):
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In which, a represented the mass of the herbs, minus moisture (g)
2.2.2. In vitro anti-calcium oxalate crystals formation assay
Condensed extracts preparation: The 45% and 96% ethanol (EtOH) extracts were
carried out by soaking technique. Samples of Terminalia catappa leaf powder (500 grams)
were soaked with 45% EtOH and 96% EtOH solvents. After 24 hours, collected the extract
and filtered it through filter paper. Repeated the procedure until the extraction was completed,
collected the extract and recovered the solvent by rotavap, collected the corresponding
condensed extract. The aqueous extract was performed by hot extraction technique. Herbal
powder (500 grams) was heated with distilled water, repeated 3 times, the liquid extract and
vacuum freeze-dry was collected, the corresponding condensed extract was collected. After
that, the condensed extract content of all samples was calculated by percentage equation.
Nucleation assay: Carried out according to Abu et al (2020) [9], with appropriate
adjustments. Accurately take 100 µL of each test sample (45% EtOH extract, 96% EtOH
extract, aqueous extract and positive control) at different concentrations into separate
eppendorfs containing 950 µL CaCl2. The crystal formation reaction started with the
addition 950 µL Na2C2O4. The reaction mixture was incubated in the dark at 37 ºC for 15
min, then optical density was measured at 620 nm wavelength. All experiments were
performed 3 times. Cystone will be used as a positive control. The percentage of inhibition
(%) was calculated according to the formula (2.3) as follows:
%inhibition = [ODblank sample – ODtest sample]/ODblank sample] x 100.
Statistical analysis: Calculate the percentage of inhibition of each condensed extract
of different concentrations. From there, determine IC50 (concentration at which the
condensed extract inhibits 50% of calcium oxalate crystals formation) of each hydrophilic
extract. In the same extracts, the OneWay-ANOVA test was used to evaluate the difference
in inhibitory effects at different concentrations.
III. RESULTS
3.1. Preliminary analysis of phytochemicals of Terminalia catappa leaves
3.1.1. Moisture
The average moisture of the samples of herbs after triplicate tests was 11.625%. This
result was lower than value defined in the Vietnamese Pharmacopoeia (13%). The moisture
was suitable for storage and use.
3.1.2. Content of condensed extracts
The results of the content of 96% EtOH, 45% EtOH and aqueous extracts were
presented in Table 1. The highest value belonged to 96% EtOH extract with 8.24%,
followed by 45% EtOH extract with 6.01% and aqueous extract with 2.99%, respectively.
Table 1. Contents (%) of condensed extracts from Terminalia catappa leaves
Extracts
Mass (g)
EtOH 96%
EtOH 45%
Aqueous
41.18
30.05
14.96
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3.1.3. Preliminary qualitative analysis of phytochemical composition
In general, aqueous extract of Terminalia catappa leaves contains noticeable chemical
components such as flavonoid, tanninoid and saponin (Table 2). These phytocompounds may
be major components affecting the biological properties of Terminalia catappa leaves.
Table 2. Phytochemical composition of aqueous extract of Terminalia catappa leaves
Natural compounds
Reagents used
Results
Alkaloid
Wagner
-
Dragendorff
-
Valse- Mayer
-
Flavonoid
FeCl3 5%
+
Cyanidin
+
Anthocyanoside
HCl 10%,
NaOH 10%
-
Proanthocyanidin
HCl 10%
-
Tanninoid
Gelatin
++
FeCl3 5%
++
Saponin
Shaked with water
++
3.1.4. Tanninoid content
Oxidation titration method (Lowenthal method)
The results of average volume values V (mL) used for titration of extracts and blank
sample of 0.1N KMnO4 were 15.26 mL and 1.13 mL, respectively. Using formula (2.1) to
calculate, the tanninoid content was 10.88%.
Skin powder method
The mass results (g) of three test sample solutions after testing were presented in
table 3. Apply formula (2.2) to calculate the tanninoid content was 10.70%.
Table 3. Mass (g) of 03 samples of test solution
Samples
Mass (g)
To
0.128
T1
0.141
T2
0.162
Through 2 methods, the average tanninoid content was estimated about 10.79%.
3.2. Inhibition effect on in vitro calcium oxalate kidney stone formation
The results of inhibition of calcium oxalate kidney stone formation in vitro on
different hydrophilic extracts from Terminalia catappa leaves were shown in Table 4.
Table 4. Inhibition effect on calcium oxalate kidney stone formation in vitro
Concentration
(μg/mL)
OD values
(% Inhibition effect)
Aqueous ex.
45% EtOH ex.
96% EtOH ex.
Cystone
100
0.0716 0.0021a
(18.26%)
0.0720 0.0027a
(17.86%)
0.0813 0.0015a
(7.21%)
0.0713 0.0032a
(18.63%)
200
0.0637 0.0015b
( 27.37%)
0.0600 0.0010b
(31.55%)
0.0720 0.0010b
(17.83%)
0.0543 0.0012b
(38.02%)
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133
Concentration
(μg/mL)
OD values
(% Inhibition effect)
Aqueous ex.
45% EtOH ex.
96% EtOH ex.
Cystone
400
0.0580 0.0017b
(33.83%)
0.0526 0.0015b
(39.92%)
0.0667 0.0006c
(23.95%)
0.0420 0.0010c
(52.09%)
600
0.0437 0.0035c
(50.20%)
0.0460 0.0036c
(47.53%)
0.0590 0.0000d
(32.69%)
0.0343 0.0031d
(60.85%)
800
0.0363 0.0021d
(58.55%)
0.0413 0.0006d
(52.84%)
0.0503 0.0012e
(42.58%)
0.0233 0.0006e
(73.39%)
1000
0.0263 0.0015e
(69.95%)
0.0357 0.0024e
(59.31%)
0.0433 0.0006f
(50.57%)
0.0153 0.0021f
(82.51%)
Values followed by different letters in the same column were statistically different at the 5% level.
Based on the percentage of inhibition effect, the regression equations were
established and the IC50 values (μg/mL) were calculated and presented in Table 5.
Table 5. Regression equation and IC50 of test samples
Samples
Regression equation
IC50 (μg/mL)
Cystone
y = 0.0007 x + 0.2039 (R2 = 0.9516)
423.05
Aqueous extract
y = 0.0006 x + 0.1384 (R2 = 0.9899)
602.67
45% EtOH extract
y = 0.0004 x + 0.1984 (R2 = 0.9362)
754.02
96% EtOH extract
y = 0.0005 x + 0.0554 (R2 = 0.9852)
889.20
IV. DISCUSSION
4.1. Preliminary analysis of phytochemical compositions
4.1.1. Preliminary phytochemical compositions
In general, aqueous extract contains chemical components such as flavonoid,
tanninoid and saponin. These data is similar to the qualitative analysis of Terminalia
catappa leaves in São Paulo, Brazil and Hanoi, Vietnam according to Katiki et. al. (2017)
[10] and H.D. Hung et. al. (2022) [11], respectively. The phytochemical compositions will
certainly influence the biological effects of herbal medicine.
4.1.2. Content of tanninoid
The result showed the contents of tanninoid in both quantitative methods were
similar. The average content was estimated about 10.8%. This result was approximately 10
times higher than that in the research of Lokman et. al. (2014) with total tanninoid content
of Terminalia catappa leaves harvested in Malaysia was 1.15% [12]. However, the used
solvent in this article was 70% acetone. Tanninoids were polar compounds, thus water was
selected for extraction in our study. Besides, another research done in Indonesia showed
that the tanninoid content in Terminalia catappa leaves was 12.45% with the solvent being
85% ethanol [13]. Hereby, it is important to select an appropriate solvent to take the highest
active ingredient content. Besides, water is a friendly solvent, and easy to be used in practice
compared to acetone or ethanol.
4.2. Inhibition of calcium oxalate crystallization in vitro
The three hydrophilic extracts of Terminalia catappa leaves show the inhibitory
activity for crystallization in the concentration range being 1000-100 μg/mL. In which, the